Interest in the anatomy of the interatrial septum (IAS) and its adjoining atrioventricular (AV) junction has risen enormously in the past two decades with the simultaneous evolution of left-sided percutaneous structural heart disease and complex electrophysiologic procedures. These procedures require, in fact, a direct route to the left atrium through the IAS. Thus, a thorough understanding of the complex anatomy of the IAS and AV junction is essential for performing a safe and effective transseptal puncture. There is a large amount of literature carefully describing the anatomy of the IAS and AV junction. These studies are based almost exclusively on anatomic specimens. Conversely, in this review the authors emphasize the role of noninvasive imaging techniques, in particular cardiac magnetic resonance, two- and three-dimensional transesophageal echocardiography, and computed tomography in visualizing specific aspects of the normal IAS and AV junction. Where appropriate, the authors present images side by side, with corresponding anatomic specimens.